Transfection is the process of introducing nucleic acids into eukaryotic cells by non-viral methods. This gene transfer technology uses various physical or chemical methods to study gene function and protein expression in the context of a cell. Transfection applications have increased due to the development of reporter gene systems and selection methods for stable maintenance and expression of transferred DNA. The availability of transfection reagents together with assay-based reporter technology allows for the study of mammalian promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein-protein interactions, translation and recombinant events.
Transfection methods allow the introduction of negatively charged molecules (e.g. phosphate backbones of DNA and RNA) into cells having a negatively charged membrane. Physical methods like microinjection or electroporation simply punch through the membrane and introduce the DNA directly into the cytoplasm. Chemicals such as calcium phosphate and DEAE-dextran, or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule. The DNA-transfection reagent complex easily crosses the cell membrane, especially for lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer of the cell.
There is a plethora of transfection reagents based on a cationic lipid that have been used to deliver large anionic molecules, such as nucleic acids, into certain types of cells (see Feigner et al., Nature 337:387-388 (1989); Proc. Natl. Acad. Sci. USA 84:7413 (1987); Behr et al., Proc. Natl. Acad. Sci. USA 86: 6982 (1989)). Both mono- and polycationic lipids have been used alone or in combination with other reagents, for delivery of these macromolecules. A large number of cationic lipids are commercially available. These agents are not, however, universally effective in all cell types and have the added drawback of being toxic. In many cases, the cationic lipids have to be used in combination with other reagents such as peptides to be effective in transfection. Moreover, these reagents require relatively complex protocols and are inconvenient to use.
It is apparent; therefore, that there is still a need for transfection reagents that are less toxic than the existing reagents and are universally effective. In particular, improved methods and transfection reagents are needed for introducing nucleic acids into primary cells as well as for the delivery of particular nucleic acids such as siRNA into cells.